Two key polymorphisms in a newly discovered allele of the Vitis vinifera TPS24 gene are responsible for the production of the rotundone precursor α-guaiene

Rotundone was initially identified as a grape-derived compound responsible for the peppery aroma of Shiraz wine varieties. It has subsequently been found in black and white pepper and several other spices. Because of its potent aroma, the molecular basis for rotundone formation is of particular relevance to grape and wine scientists and industry. We have identified and functionally characterized in planta a sesquiterpene synthase, VvGuaS, from developing grape berries, and have demonstrated that it produces the precursor of rotundone, α-guaiene, as its main product. The VvGuaS enzyme is a novel allele of the sesquiterpene synthase gene, VvTPS24, which has previously been reported to encode VvPNSeInt, an enzyme that produces a variety of selinene-type sesquiterpenes. This newly discovered VvTPS24 allele encodes an enzyme 99.5% identical to VvPNSeInt, with the differences comprising just 6 out of the 561 amino acid residues. Molecular modelling of the enzymes revealed that two of these residues, T414 and V530, are located in the active site of VvGuaS within 4 Å of the binding-site of the substrate, farnesyl pyrophosphate. Mutation of these two residues of VvGuaS into the corresponding polymorphisms in VvPNSeInt results in a complete functional conversion of one enzyme into the other, while mutation of each residue individually produces an intermediate change in the product profile. We have therefore demonstrated that VvGuaS, an enzyme responsible for production of the rotundone precursor, α-guaiene, is encoded by a novel allele of the previously characterized grapevine gene VvTPS24 and that two specific polymorphisms are responsible for functional differences between VvTPS24 alleles

BHPR research: qualitative1. Complex reasoning determines patients' perception of outcome following foot surgery in rheumatoid arhtritis

Background: Foot surgery is common in patients with RA but research into surgical outcomes is limited and conceptually flawed as current outcome measures lack face validity: to date no one has asked patients what is important to them. This study aimed to determine which factors are important to patients when evaluating the success of foot surgery in RA Methods: Semi structured interviews of RA patients who had undergone foot surgery were conducted and transcribed verbatim. Thematic analysis of interviews was conducted to explore issues that were important to patients. Results: 11 RA patients (9 ♂, mean age 59, dis dur = 22yrs, mean of 3 yrs post op) with mixed experiences of foot surgery were interviewed. Patients interpreted outcome in respect to a multitude of factors, frequently positive change in one aspect contrasted with negative opinions about another. Overall, four major themes emerged. Function: Functional ability & participation in valued activities were very important to patients. Walking ability was a key concern but patients interpreted levels of activity in light of other aspects of their disease, reflecting on change in functional ability more than overall level. Positive feelings of improved mobility were often moderated by negative self perception ("I mean, I still walk like a waddling duck”). Appearance: Appearance was important to almost all patients but perhaps the most complex theme of all. Physical appearance, foot shape, and footwear were closely interlinked, yet patients saw these as distinct separate concepts. Patients need to legitimize these feelings was clear and they frequently entered into a defensive repertoire ("it's not cosmetic surgery; it's something that's more important than that, you know?”). Clinician opinion: Surgeons' post operative evaluation of the procedure was very influential. The impact of this appraisal continued to affect patients' lasting impression irrespective of how the outcome compared to their initial goals ("when he'd done it ... he said that hasn't worked as good as he'd wanted to ... but the pain has gone”). Pain: Whilst pain was important to almost all patients, it appeared to be less important than the other themes. Pain was predominately raised when it influenced other themes, such as function; many still felt the need to legitimize their foot pain in order for health professionals to take it seriously ("in the end I went to my GP because it had happened a few times and I went to an orthopaedic surgeon who was quite dismissive of it, it was like what are you complaining about”). Conclusions: Patients interpret the outcome of foot surgery using a multitude of interrelated factors, particularly functional ability, appearance and surgeons' appraisal of the procedure. While pain was often noted, this appeared less important than other factors in the overall outcome of the surgery. Future research into foot surgery should incorporate the complexity of how patients determine their outcome Disclosure statement: All authors have declared no conflicts of interes

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Adding 6 months of androgen deprivation therapy to postoperative radiotherapy for prostate cancer: a comparison of short-course versus no androgen deprivation therapy in the RADICALS-HD randomised controlled trial

Background Previous evidence indicates that adjuvant, short-course androgen deprivation therapy (ADT) improves metastasis-free survival when given with primary radiotherapy for intermediate-risk and high-risk localised prostate cancer. However, the value of ADT with postoperative radiotherapy after radical prostatectomy is unclear. Methods RADICALS-HD was an international randomised controlled trial to test the efficacy of ADT used in combination with postoperative radiotherapy for prostate cancer. Key eligibility criteria were indication for radiotherapy after radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to radiotherapy alone (no ADT) or radiotherapy with 6 months of ADT (short-course ADT), using monthly subcutaneous gonadotropin-releasing hormone analogue injections, daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as distant metastasis arising from prostate cancer or death from any cause. Standard survival analysis methods were used, accounting for randomisation stratification factors. The trial had 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 80% to 86% (hazard ratio [HR] 0·67). Analyses followed the intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov, NCT00541047. Findings Between Nov 22, 2007, and June 29, 2015, 1480 patients (median age 66 years [IQR 61–69]) were randomly assigned to receive no ADT (n=737) or short-course ADT (n=743) in addition to postoperative radiotherapy at 121 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 9·0 years (IQR 7·1–10·1), metastasis-free survival events were reported for 268 participants (142 in the no ADT group and 126 in the short-course ADT group; HR 0·886 [95% CI 0·688–1·140], p=0·35). 10-year metastasis-free survival was 79·2% (95% CI 75·4–82·5) in the no ADT group and 80·4% (76·6–83·6) in the short-course ADT group. Toxicity of grade 3 or higher was reported for 121 (17%) of 737 participants in the no ADT group and 100 (14%) of 743 in the short-course ADT group (p=0·15), with no treatment-related deaths. Interpretation Metastatic disease is uncommon following postoperative bed radiotherapy after radical prostatectomy. Adding 6 months of ADT to this radiotherapy did not improve metastasis-free survival compared with no ADT. These findings do not support the use of short-course ADT with postoperative radiotherapy in this patient population

Duration of androgen deprivation therapy with postoperative radiotherapy for prostate cancer: a comparison of long-course versus short-course androgen deprivation therapy in the RADICALS-HD randomised trial

Background Previous evidence supports androgen deprivation therapy (ADT) with primary radiotherapy as initial treatment for intermediate-risk and high-risk localised prostate cancer. However, the use and optimal duration of ADT with postoperative radiotherapy after radical prostatectomy remains uncertain. Methods RADICALS-HD was a randomised controlled trial of ADT duration within the RADICALS protocol. Here, we report on the comparison of short-course versus long-course ADT. Key eligibility criteria were indication for radiotherapy after previous radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to add 6 months of ADT (short-course ADT) or 24 months of ADT (long-course ADT) to radiotherapy, using subcutaneous gonadotrophin-releasing hormone analogue (monthly in the short-course ADT group and 3-monthly in the long-course ADT group), daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as metastasis arising from prostate cancer or death from any cause. The comparison had more than 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 75% to 81% (hazard ratio [HR] 0·72). Standard time-to-event analyses were used. Analyses followed intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov , NCT00541047 . Findings Between Jan 30, 2008, and July 7, 2015, 1523 patients (median age 65 years, IQR 60–69) were randomly assigned to receive short-course ADT (n=761) or long-course ADT (n=762) in addition to postoperative radiotherapy at 138 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 8·9 years (7·0–10·0), 313 metastasis-free survival events were reported overall (174 in the short-course ADT group and 139 in the long-course ADT group; HR 0·773 [95% CI 0·612–0·975]; p=0·029). 10-year metastasis-free survival was 71·9% (95% CI 67·6–75·7) in the short-course ADT group and 78·1% (74·2–81·5) in the long-course ADT group. Toxicity of grade 3 or higher was reported for 105 (14%) of 753 participants in the short-course ADT group and 142 (19%) of 757 participants in the long-course ADT group (p=0·025), with no treatment-related deaths. Interpretation Compared with adding 6 months of ADT, adding 24 months of ADT improved metastasis-free survival in people receiving postoperative radiotherapy. For individuals who can accept the additional duration of adverse effects, long-course ADT should be offered with postoperative radiotherapy. Funding Cancer Research UK, UK Research and Innovation (formerly Medical Research Council), and Canadian Cancer Society

Characterisation of PpMDHARs and PpENA1 from the moss, Physcomitrella patens.

Identifying a genetic basis for the tolerance to salinity exhibited by the resilient moss, Physcomitrella patens, could provide valuable information for use in the selection or modification of salinity tolerance in crop plants. The overall aim of the work described in this thesis was to identify, express and functionally characterise the protein products of two putative salinity tolerance genes from Physcomitrella, namely PpMdhar and PpENA1. The characterisation of PpMdhar and PpENA1 represents a two-pronged approach into investigating the salinity tolerance of Physcomitrella at the biochemical and transport level, respectively. The enzymes encoded by PpMdhars, monodehydroascorbate reductases (MDHARs), are central to the ascorbate-glutathione cycle, and recycle monodehydroascorbate molecules into the antioxidant, ascorbate. Hence, MDHARs play a part in maintaining the capacity of plant cells to remove toxic reactive oxygen species. Given that the production of reactive oxygen species is greatly increased in plants under salt stress, and that Physcomitrella is tolerant of high salt, MDHAR enzymes were expressed to determine whether they exhibit increased enzymic activity when compared with MDHARs from higher plants. The protein encoded by PpENA1 is Na⁺ transporting ATPase, which actively transports toxic Na⁺ ions across the cell membranes, and thereby minimizes the level of Na⁺ that accumulates in the cytoplasm. Thus, in contrast to the mechanism by which MDHARs may help Physcomitrella deal with the secondary effects of high salt, the PpENA1 protein could enable the moss to actively exclude Na⁺ ions, and thereby avoid cellular toxicity. A link between salinity and the transcription of PpMdhar and PpENA1 is reported here, and the function of each gene is investigated. A comprehensive characterisation of the enzymic action of expressed PpMDHAR enzymes is described, demonstrating that the biochemical mechanisms used by Physcomitrella in dealing with salt-induced reactive oxygen species are likely to be conserved with vascular plants. The physiological effects of the expression of PpENA1 are investigated via complementation experiments in yeast, and the membrane location of the protein is determined. The Na⁺ binding-sites of PpENA1 are predicted using homology modelling and amino acid residues crucial for Na⁺ transport are tested experimentally via site-directed mutagenesis. Finally, the introduction of a new, functional Na⁺ binding-site into an inactivated form of the PpENA1 protein demonstrates that a degree of control is possible over the Na⁺ binding-sites in PpENA1.Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 200